2013: Update on antimicrobial therapy in mare reproduction Update on antimicrobial therapy in mare reproduction Kristina G. Lu Hagyard Equine Medical Institute, Lexington, KY Bacterial uterine infections are associated with significant time and monetary loss in the equine breeding industry. The incidence of identified bacterial uterine infection is estimated at 25 to 60% of barren mares.1 Treatment and resolution of mare urogenital infection involves a holistic approach which must include consideration of the systemic health of the mare, correction of urogenital anatomic defects, enhancement of uterine clearance in non-pregnant mares, identification of specific infections, and appropriate antimicrobial selection and administration. The following is a brief review of antimicrobial properties and classes, and discussion regarding antimicrobial selection for use in specific mare reproductive disease states. Excellent additional resources are available.2 In this age of increasing bacterial and fungal antimicrobial resistance and heightened awareness of antimicrobial use in animals, judicious and wise employment of antimicrobials should be emphasized. Keywords: Antibiotics, antifungals, chelators, endometritis, placentitis General antimicrobial properties Bactericidal antimicrobials a) Time dependent (there is no benefit in increasing the drug concentration above the minimum inhibitory concentration (MIC); instead the time the drug concentration is above MIC is most important); examples are beta-lactams and trimethoprim-sulfonamide b) Concentration dependent (rate of bacterial killing increases as the peak concentration increases above the pathogen’s MIC rather than the duration of the drug’s concentration); examples are aminoglycosides, fluoroquinolones, metronidazole, and peptides Bacteriostatic antimicrobials Concentration dependent; examples are macrolides, tetracyclines, and chloramphenicol Antimicrobial classes Beta-lactam: penicillin Commonly used formulations are procaine penicillin, aqueous penicillin salts, ampicillin, ticarcillin; those less commonly used in equine reproduction include carbapenems (imipenem, meropenem) Mechanism of action. Beta-lactams are time-dependent antimicrobials that interfere with cell wall formation by inhibiting the penicillin-binding proteins that catalyze polymer cross-linking necessary for cell wall formation. Beta-lactams are bactericidal for replicating cells, thus clinical benefit is reduced when used concurrently with a bacteriostatic drug. Penicillins are generally most effective against gram- positive organisms, however they vary in efficacy against gram-negative organisms. Differences in organism susceptibility are attributable to differences in penicillin-binding protein receptor sites, the amount of peptidoglycan present in the cell wall, drug penetration of the outer cell membrane of gram- negative bacteria, and beta-lactamase-induced resistance. Distribution. Penicillins are well distributed throughout the body, reaching MIC in most organs including the reproductive tract. They are ionized, poorly lipid soluble, and tend to cross biological membranes poorly. They are excreted almost entirely by the kidney and thus can be useful to treat urinary tract infections caused by sensitive organisms. 503 Clinical Theriogenology • Volume 5, Number 4 • December 2013 Synergism. Beta lactams are synergistic with aminoglycosides Evaluation of endometrial ampicillin concentration 24 hours after a three-gram intrauterine infusion of ampicillin revealed drug concentrations above MIC for investigated isolates 24 hours after infusion.3 Intrauterine infusion of 6.2-g ticarcillin/clavulanic acid resulted in endometrial tissue concentrations of 150-424 micrograms/g 60 minutes after infusion. Clearance is rapid, potentially necessitating frequent administration to maintain concentrations.4 Systemically administered potassium penicillin achieved MIC for Streptococcus equi subspecies zooepidemicus (hereafter S. zooepidemicus) in allantoic fluid.5 Penicillins are also used for treatment of leptospirosis. Addition of Timentin® (ticarcillin plus clavulanic acid) to Inra96® semen extender to a final concentration of 0.5 to 1.5 mg/mL can limit the growth of Taylorella equigenitalis.6 Addition of Timentin® to semen extender (1 mg/mL) did not impair sperm quality but did not provide added protection against bacteria commonly found in equine semen.7 Beta-lactams: cephalosporins Commonly used formulations are ceftiofur sodium, ceftiofur crystalline free acid, ceftiofur hydrochloride Mechanism of action: Cephalosporins are also beta-lactam antimicrobials, however the molecular structure of the cephalosporin group of antimicrobials make them inherently more resistant to beta- lactamases. Cephalosporins are grouped in their order of development. Ceftiofur is a third generation cephalosporin in common usage in equine medicine. Ceftiofur is rapidly metabolized by plasma esterases to the active metabolite, desfuroylceftiofur. Distribution. Widespread distribution into tissues in extracellular fluid is achieved however passage across membranes and physiological barriers is poor. Excretion is largely through the urinary tract. Uses in reproduction. In mares administered ceftiofur sodium (2 mg/kg IM BID), ceftiofur sodium was not detected in endometrial tissue after the fifth dose when steady state concentrations were achieved.8 In mares administered ceftiofur hydrochloride (2.2 mg/kg IM), a ceftiofur derivative was detected in endometrial tissue 24 hours after administration at concentrations above reported MIC for S. zooepidemicus and Escherichia coli.9 In mares administered ceftiofur crystalline free acid (6.6 mg/kg IM), endometrial drug concentrations remained above MIC for S. zooepidemicus for 96 hours.10 In a study evaluating efficacy of ceftiofur crystalline free acid in equine placentitis, the drug was not found in effective concentrations in the placenta, fetal tissue, amnionic fluid or fetal serum and did not improve foal survival rates.11 Intrauterine ceftiofur sodium (1 g in 100 mL saline) has been found to be safe and effective.12 Beta-lactamase inhibitors Clavulanic acid, a broad-spectrum beta-lactamase inhibitor, is used in combination with other antimicrobials such as ticarcillin. Certain bacteria can resist the action of beta-lactam antimicrobials by producing beta-lactamases, enzymes that degrade beta-lactam antimicrobials by opening the beta-lactam ring. Genes encoding beta-lactamase are transmitted through bacterial populations with plasmids and transposons as an important mechanism of spreading resistance. Beta-lactamase inhibitors bind irreversibly to beta-lactamases allowing the accompanying beta-lactam antibiotic to bind to the penicillin binding protein. Peptide antibiotics A commonly used formulationis polymyxin B 504Clinical Theriogenology • Volume 5, Number 4 • December 2013 Mechanism of action. Polymyxins are peptide antibiotic products of Bacillus polymyxa. Polymyxin B is toxic at systemic antimicrobial doses with nephrotoxic, neurotoxic, and neuromuscular blocking effects, thus it is used topically for its antimicrobial properties and used systemically at sub- antimicrobial doses for binding and inactivating endotoxin (lipopolysaccharide). When used as an antimicrobial, polymyxin B is bactericidal and concentration-dependent. This cationic peptide disrupts the outer membrane of gram-negative bacteria by binding lipopolysaccharide (LPS), and increasing cell permeability. Gram-positive bacteria are resistant. Distribution. Polymyxin diffuses poorly through biologic membranes and attains low concentrations in transcellular fluids. Uses in reproduction. Polymyxin B can be infused into the uterus for gram-negative bacterial infections and used systemically for endotoxemia as may be seen in post-foaling complications. Macrolides Macrolides (erythromycin, azithromycin, clarithromycin) are predominantly bacteriostatic antimicrobials that inhibit protein synthesis by reversibly binding to the 50S ribosomal subunit, and thus are specific for prokaryotic cells. In adult horses, macrolide use can be associated with severe diarrhea. Macrolides are infrequently used in mare reproduction and will certainly be highlighted in discussion on antimicrobial use in foals and weanlings. Aminoglycosides Commonly used formulations are entamicin and amikacin; others include kanamycin, neomycin, streptomycin, dihydrostreptomycin, tobramycin. Mechanism of action. Aminoglycosides are bactericidal, concentration dependent antimicrobials used primarily to treat aerobic gram-negative bacteria and staphylococci. Their bactericidal action is influenced by pH, being most active in alkaline pH. Aminoglycosides must penetrate bacterial cell walls, with penetration enhanced when in combination with drugs that interfere with the cell wall (e.g., penicillins). Once within the bacteria, they bind to the 30s ribosomal subunit (prokaryotic) and inhibit bacterial protein synthesis. The most clinically important resistance is due to enzymes from plasmids that are transferrable between bacteria. Elimination is by renal excretion (glomerular filtration) and nephrotoxicity is an important side effect. Risk factors for aminoglycoside toxicity include prolonged therapy (>7-10 days), multiple doses per day, acidosis, electrolyte disturbances, dehydration, concurrent nephrotoxic drug therapy, age, pre-existing renal disease, and elevated plasma trough concentrations. Ototoxicity may also occur. Amikacin has the broadest spectrum of the aminoglycosides, and is the least nephrotoxic. Concurrent administration of gentamicin with phenylbutazone was found to decrease elimination half-life of gentamicin by 23% and decrease the volume of distribution by 26% while pharmacokinetics of phenylbutazone were not affected. Aminoglycosides are pharmaceutically incompatible with many beta-lactams and should not be mixed in same syringe.2 Distribution. Aminoglycosides are large polycationic molecules that are poorly lipid soluble, with limited ability to enter cells and penetrate cellular barriers unaided. Purulent or necrotic debris bind and inactivate aminoglycosides. Synergism. Aminoglycosides are synergistic with beta-lactams and trimethoprim-sulfonamide. Antagonism. Antagonism may occur with chloramphenicol, tetracycline, erythromycin; should not be mixed with penicillins in vitro.2 505 Clinical Theriogenology • Volume 5, Number 4 • December 2013 Uses in reproduction. Intravenous gentamicin (6.6 mg/kg IV SID) yielded concentrations in allantoic fluid above MIC for potential gram-negative pathogens such as E. coli.5 Amikacin is labeled for intrauterine use at a dose 2 g in 200 ml saline q 24 hrs for three consecutive days for endometritis, metritis, and pyometra in mares. Gentamicin (2 g in 80 mL saline) intrauterine infusion has been associated with shorter duration histologic inflammation though more cellular changes were seen with scanning electron microscopy compared to saline controls.13,14 It is generally recommended that gentamicin be buffered or well diluted in saline to minimize irritation. Tetracyclines Commonly used formulations are oxytetracycline and doxycycline Mechanism of action. Tetracyclines are broad spectrum, bacteristatic antimicrobials that diffuse through the outer cell membrane to reversibly bind to the 30s ribosomal subunit to inhibit protein synthesis. They are strong chelating agents with the ability to chelate divalent and trivalent ions such as calcium, with potential to cause tooth discoloration. There is widespread acquired resistance particularly among gram-negative bacteria and variable susceptibility in many Staphylococcus sp., Streptococcus sp., Enterococcus sp., E. coli, and Klebsiella sp. Resistance is rare in obligate intracellular pathogens such as Ehrlichia sp. and Anaplasma sp. Doxycycline (semi-synthetic) is more lipid soluble than oxytetracycline. Tetracyclines are relatively safe, with reported risks of enterocolitis, occasional fatal anaphylaxis, and collapse if intravenous administration is not slow. Renal tubular damage may occur if outdated product used. Distribution. Tetracyclines are widely distributed. Synergism. Tetracyclines are synergistic with polymyxins by enhancing bacterial uptake Uses in reproduction. After intragastric administration of doxycycline (five doses, 10 mg/kg BID), the endometrial concentration was approximately 1.3 micrograms/mL, above the MIC for the evaluated strains of S. zooepidemicus and Staphylococcus aureus.15 Tetracyclines can be used systemically for treatment of leptospirosis. In a study evaluating the efficacy and safety of intrauterine oxytetracycline, six grams of oxytetracycline was infused daily for three days. Endometrial oxytetracycline concentration remained above MIC for eight hours after infusion for S. zooepidemicus, Klebsiella pneumonia, Pseudomonas aeruginosa, and E. coli, with observation of transient endometrial inflammation, and thus the recommendation for further evaluation prior to routine intrauterine use.16 Chloramphenicol Mechanism of action. Chloramphenicol is a bacteriostatic, lipid soluble, neutral, broad spectrum antimicrobial. It irreversibly binds to the 50S subunit of bacterial ribosomes and also inhibits mitochondrial protein synthesis is mammalian bone marrow cells in a dose-dependent manner. Resistance occurs via inactivation by chloramphenicol acetyltransferases (CATs) shared via plasmids, transposons, and integrons. Elimination occurs through the liver. Chloramphenicol is associated with idiosyncratic aplastic anemia in humans and is restricted in some countries because of bacterial resistance and risk of aplastic anemia. Distribution. Chloramphenicol is widely distributed. Uses in reproduction. Anecdotally used for intrauterine infusion when proteinaceous debris is present (W. Zent, personal comminication). Systemic use is based on clinical necessity. Sulfonamides 506Clinical Theriogenology • Volume 5, Number 4 • December 2013 Commonly used formulations are sulfadiazine, sulfasalazine, and sulfamethoxazole Mechanism of action. Sulfonamides are bacteriostatic when used alone and bactericidal when combined with trimethoprim. They interfere with biosynthesis of folic acid in bacterial cells by competitively preventing para-aminobenzoic acid (PABA) incorporation into folic acid molecule by competing with PABA for dihydropteroate synthetase. Bacteriostatic action depends on the requirement for folic acid; susceptible microorganisms must synthesize folic acid while mammalian cells use preformed folic acid. Tissue exudate or necrotic tissue must be removed for penetration. Bacterial resistance is extensive. Elimination occurs through a combination of renal excretion and metabolic transformation. Adverse side effects, though uncommon, may include urinary tract disturbances, hematopoietic disorders, dermatologic disorders, and keratoconjunctivitis sicca in dogs. Distribution. Sulfonamides are widely distributed. Uses in reproduction. Systemic administration of trimethoprim sulfamethoxazole (30 mg/kg PO BID) in combination with pentoxifylline and altrenogest have been well evaluated in placentitis models (see below). Diaminopyrimidines A commonly used formulation is trimethoprim in conjunction with sulfonamides. Mechanism of action. Trimethoprim interferes with folic acid production by inhibition of dihydrofolate reductase with greater affinity for bacterial versus mammalian enzymes, preventing synthesis of purines and thus DNA. They are bacteriostatic when used alone. Efficacy is decreased with plasmid associated resistance, and antagonism by tissue debris. Fluoroquinolones Commonly used formulations are enrofloxacin and ciprofloxacin. Mechanism of action. Fluoroquinolones are concentration dependent, bactericidal antimicrobials that bind DNA-gyrase-DNA complexes to impair negative supercoiling and target DNA topoisomerase, disrupting replication. They have activity against a wide range of gram-negative bacteria, with ciprofloxacin being most potent against Pseudomonas sp. The drug is concentrated within phagocytic cells and largely excreted in urine. Protein synthesis inhibitors such as chloramphenicol and RNA synthesis inhibitors such as rifampin may reduce efficacy. Resistance is not thought to be plasmid associated. Enrofloxacin inhibits cell proliferation, induces morphological changes and alters equine tendon structure, more pronounced in juvenile tendon cells. Arthropathies have been documented in two-week old foals, though not in adults. Fluoroquinolones are not recommended in pregnant animals, though they have been used in a pregnant mare with no known detrimental effect on the foal.2 Transient neurologic signs have been seen with rapid administration. Distribution. Fluoroquinolones are widely distributed. Synergism. Fluoroquinolones are synergistic with beta-lactams, aminoglycosides, and vancomycin. Antagonism. In vitro antagonism is seen between ciprofloxacin and chloramphenicol and ciprofloxacin and rifampin. 507 Clinical Theriogenology • Volume 5, Number 4 • December 2013 Uses in reproduction. Systemic enrofloxacin (5 mg/kg IV) achieved sufficient endometrial tissue concentrations to be used to treat endometritis caused by susceptible bacteria.17 Enrofloxacin (5 mg/kg IV) has also been proposed as a therapeutic strategy for the prevention of endometritis is susceptible mares with a recommended dose (5 mg/kg IV) pre-breeding followed by two further doses 36 to 48 hours post-breeding.18 Systemic ciprofloxacin (2.5 g PO SID) and probenecid (1 g PO SID) has been reported to effective against Pseudomonas infection.19 Intrauterine infusion of enrofloxacin has been evaluated with one author reporting enrofloxacin concentrations remaining above MIC for susceptible bacteria 24 hours after infusion of 2.5 mg/kg enrofloxacin.20 A more recent study found endometrial biopsy grades worsening from Kenney Doig grade I to grade III over a 60 day period following intrauterine infusion of 2.5 mg/kg enrofloxacin daily for three days and concluded that enrofloxacin is not suitable for conventional intrauterine infusion treatment in mares.21 Nitroimidazole A commonly used formulation is metronidazole. Mechanism of action. Metronidazole undergoes reduction of the nitro group to yield unstable intermediates some of which interact with bacterial or protozoal DNA; the reduction occurs under anaerobic conditions. Resistance is rare among usually susceptible bacteria. Anorexia can be seen with oral use. Occasional neurological signs have been reported in other species with recovery facilitated by diazepam. Distribution. Metronidazole is rapidly and well absorbed and lipophilic. It is metabolized in the liver and eliminated in the urine and manure. Hepatic metabolism may be decreased when concurrent with cimetidine, thus delaying elimination. Uses in reproduction. Uncommonly used unless an anaerobic infection in suspected. After systemic administration of metronidazole (15 mg/kg loading followed by 7.5 mg/kg q6h via nasogastric tube), the mean endometrial concentration of metronidazole was approximately 0.9 micrograms/mL.22 Rifamycins A commonly used formulation is rifampin. Mechanism of action. Rifampin is bactericidal, broad spectrum, and active against both extracellular and intracellular pathogens. It inhibits DNA-dependent RNA polymerase. Because of rapid development of resistance, rifampin is usually administered in conjunction with other antimicrobials. Distribution. Rifampin is very lipophilic and penetrates most tissues including abscesses, bone, milk and the central nervous system. Rifampin crosses the placenta and is teratogenic in rodents. Rifampin is eliminated via the liver. Uses in reproduction. Uncommonly used in reproduction but can be useful in reproductive tract associated abscesses (e.g. abscessed broad ligament hematomas or retroperitoneal abscesses). Antifungals The main sites of action of antifungal drugs are the cytoplasmic membrane (polyenes, azoles), the cell wall, and DNA or protein synthesis. Polyenes Commonly used formulations are amphotericin B, natamycin, and nystatin. 508Clinical Theriogenology • Volume 5, Number 4 • December 2013 Mechanism of action. Polyenes bind ergosterol, the principal sterol of the fungal cell membrane, causing leakage of cell contents. They also bind cholesterol in mammalian cell membranes (less avidly) which makes them the most toxic of clinically used antifungals. Polyenes are associated with renal toxicity. Amphotericin B is the mainstay for systemic fungicidal treatment of filamentous fungal infection. It is poorly soluble in water, unstable at 37°C, with maximal antifungal effects at pH 6-7.5, and decreased effects at low pH. Amphotericin B is not well absorbed orally and is thus intravenously administered. Nystatin has greater nephrotoxicity and is thus used topically (e.g. intrauterine). Uses in reproduction. Polyenes are a reasonable first choice for treatment of fungal endometritis caused by yeast. Molds are most susceptible to polyenes and less susceptible to imidazoles and triazoles.23 Azoles Commonly used formulations are imidazoles (clotrimazole, ketoconazole, and miconazole) and triazoles (itraconazole, fluconazole, and voriconazole). Mechanism of action. Azoles inhibit cytochrome p450 dependent ergosterol synthesis leading to disruption of fungal membranes. Azoles are fungistatic, but fungicidal at high concentrations. Ketoconazole is poorly water soluble, lipophilic, requires acid pH, requires nasogastric tube administration (if administered in conjunction with HCl as recommended, the suspension can be irritating to the oral cavity and throat). Oral administration at 30 mg/kg does not result in detectable serum concentrations.2 Itraconazole is poorly water soluble, lipophilic, and requires acid pH for absorption. Fluconazole is water soluble, oral absorption is unaffected by acid and it is well absorbed after oral administration, it distributes widely to tissues with the half-life in horses ~40 hours, and with 100% oral bioavailability. It can be administered orally or intravenously, though resistance is more frequent. Voriconazole is a second generation triazole, with a wide spectrum, can be administered orally or intravenously, is metabolized by liver, with excellent tissue penetration, though with limited experience in horses. Topical azoles Commonly used formulations are clotrimazole and miconazole. Uses in reproduction. Ketoconazole and clotrimazole are reasonable empiric treatment choices for uterine yeast infections based on a susceptibility survey. Fluconazole is popular due to its administration flexibility, however organisms’ resistance to this drug appears to be increasing.23 Ketoconazole is not recommended in pregnant animals. Iodine Addition of povidone-iodine to infusions or uterine lavage solutions is common for management of microbial infections and is included here for completion rather than an update. One study found that intrauterine infusion of 1% povidone-iodine solution in mares causes chronic inflammatory changes in the endometrium.24 A concentration of 0.5% povidone-iodine still demonstrated suppression of bacterial growth and is a commonly recommended ‘safer’ dose.25 Chelators Chelators increase the permeability of the bacterial cell wall and cell membrane and are synergistic with antimicrobials including gentamicin, penicillin, oxytetracycline and chloramphenicol. In general, first and second generation chelators have a greater application in treating gram-negative infections. The third generation chelator, Tricide®, appears to potentiate the effects of antimicrobials against gram-positive and gram-negative bacteria as well as yeast and fungi.26 509 Clinical Theriogenology • Volume 5, Number 4 • December 2013 Topical antimicrobials Topical antimicrobials are important components of therapy for vaginal, vestibular and vulvar inflammation and necrosis as most commonly occurs post-foaling. Additionally, the clitoris can occasionally be found to harbor pathogenic organisms and maintenance of the mare’s uterine health may benefit from cleansing the clitoris. Ointments that have anecdotal utility include nystatin, neomycin sulfate, thiostrepton and triamcinolone acetonide ointment (Animax®), bovine intramammary treatments (cephapirin), and sodium hypochlorite hydrogel (Anasept®). Diagnostic methods for identifying infection The use of intrauterine or systemic antimicrobials for endometritis, the most common mare reproductive tract infection, is ideally preceded by organism identification and antimicrobial sensitivity. Methods for diagnosing endometritis include a uterine swab for microbial culture, endometrial cytology to evaluate for the presence of inflammation, and an endometrial biopsy for histopathology and/or microbial culture. In a comparison of the uterine swab versus endometrial biopsy for assessment of any bacterial growth, one study found that in 2% of all positive cultures, bacterial growth was observed from the swab and not from the biopsy, while in 55% of positive cultures the swab was negative for bacterial growth and the biopsy positive. Calculation of the sensitivity and specificity was performed in two scenarios: culture using a swab compared to culture from an endometrial biopsy as the gold standard, and culture from either a swab or a biopsy compared to inflammatory cells seen in the biopsy as the gold standard.27 Swab with culture from biopsy as gold standard: Sensitivity Specificity Positive Predictive Value Negative Predictive Value Swab culture 0.44 0.98 0.95 0.74 Tests with inflammatory cells in the biopsy as the gold standard3: In an assessment of subclinical endometritis as defined by polymorphonuclear cells (PMNs) present during diestrus in the stratum compactum of the endometrial biopsy in the absence of intrauterine fluid, another study compared the value of a uterine swab, a cytology brush, and an endometrial biopsy for cytological and bacteriological diagnosis of endometritis, with the following results:28 Sensitivity Specificity Cytology Bacteriology Cytology Bacteriology Swab 0.00 0.33 0.93 0.83 Brush 0.17 0.25 0.83 0.80 Biopsy 0.25 0.25 0.85 0.95 Brush: cytology+bacteriology 0.42 0.70 These studies suggest that a uterine swab for microbial culture used alone is insufficient for diagnosis of endometritis and that diagnostic ability is improved with addition of at least endometrial cytology. Thus, if bacterial infection is suspected as an underlying cause of the inflammation, antimicrobial treatment may be empirical. Sensitivity Specificity Positive Predictive Value Negative Predictive Value Swab culture 0.34 1.00 1.00 0.44 Cytology 0.77 1.00 1.00 0.62 Biopsy culture 0.82 0.92 0.97 0.67 510Clinical Theriogenology • Volume 5, Number 4 • December 2013 Bacterial growth states It has been suggested that bacteria are able to survive for long periods in a “dormant” or “viable but non-culturable (VBNC)” state during which time neither plating onto solid media nor inoculation into liquid media lead to growth of cells. Furthermore, bacteria in dormant states are resistant to antimicrobial therapy. Testing for the presence of these bacteria requires fluorescence microscopy and fluorescence labeled antibodies, propidium iodide in conjunction with molecular probes, or amplification of bacterial mRNA using RT-PCR techniques. Some species of bacteria retain their pathogenic potential during dormancy. A human example of the medical importance of non-growth states is Mycobacterium tuberculosis.29-31 Research is actively targeting signals for bacteria to resume growth. An example of such a signal is the resuscitation-promoting factor (Rpf), an enzyme that has been shown to increase the culturability of dormant bacteria. A mechanism by which this enzyme functions in conjunction with other bacterial proteins is under investigation.32 In a study to evaluate for a similar bacterial dormancy state in the equine endometrium, Petersen et al used fluorescence in situ hybridization (FISH) to evaluate S. zooepidemicus endometritis in mares treated with systemic antibiotics. Despite systemic antibiotic treatment, streptococci could still be visualized deep within the endometrium.33 Research into the effect of a proprietary “activation” signal for resumption of bacterial growth is ongoing.34 Post-mating induced endometritis Treatment of post-mating induced endometritis in the form of routine antimicrobial infusion post- breeding is common in the Thoroughbred breeding industry. As in any healthy debate, there are at least two sides to the argument. On one hand, there is limited published, statistically significant evidence supporting this practice.35 Copious use of antimicrobials is also scrutinized for potential antimicrobial resistance development. On the other hand, a study evaluating Thoroughbred reproductive efficiency and financial value found that mares that were barren twice over a seven-year investment were not profitable. This taken together with drift (in the same study, drift was 13.4 +/- 23.2 days) demonstrates that the average mare needs to become pregnant within one to two estrous cycles every year.36 This may drive the broad treatment for even potential post-breeding infectious endometritis. In a recent study of S. zooepidemicus from horses conducted by the University of Kentucky Veterinary Diagnostic Laboratory, resistance of S. zooepidemicus has not developed.37 Placentitis Thirty-four percent of equine abortions or stillbirths were associated with feto-placental infection. Of these, 17.8% had an identified bacterial etiology.38 Antimicrobials are a critical component of bacterial placentitis treatment. Antimicrobials that have evidence of reaching allantoic fluid include penicillin G (22,000 units/kg IM BID), gentamicin (6.6 mg/kg IV SID) and trimethoprim sulfamethoxazole (15-30 mg/kg PO BID).39,40 Other antimicrobials are anecdotally used with effect. In an experimental ascending placentitis model with beta-Streptococcus, the introduced organism was be cultured from the uterus post-foaling in both treated and untreated controls. The treated ponies received antimicrobials (trimethoprim sulfamethoxazole 30 mg/kg PO BID) continuously until parturition.41 This information suggests that antimicrobial therapy in pregnant mares with placentitis may need to be prolonged and weighed against side effects of long-term antimicrobial therapy. References 1. Causey RC, Weber JA, Emmans EE, et al: The equine immune response to Streptococcus equi subspecies zooepidemicus during uterine infection. Vet J 2006;172:248-257. 2. Giguere S, Prescott JF, Baggot JD, et al, editors: Antimicrobial therapy in veterinary medicine. 4th ed. Ames (IA): Blackwell Publishing; 2006. 3. Love CC, Strzemienski PJ, Kenney RM: Endometrial concentrations of ampicillin in mares after intrauterine infusion of the drug. Am J Vet Res 1990;51:197-199. 511 Clinical Theriogenology • Volume 5, Number 4 • December 2013 4. Van Camp SD, Papich MG, Whitacre MD: Administration of ticarcillin in combination with clavulanic acid intravenously and intrauterinely to clinically normal oestrous mares. J Vet Pharmacol Ther 2000;23:373-378. 5. Murchie T, Macpherson ML, LeBlanc M, et al: A microdialysis model to detect drugs in the allantoic fluid of pregnant pony mares. Proc Annu Conv Am Assoc Equine Pract; 2003. p. 118-121. 6. Olivieri BT, Love BC, Rezabek GB, et al: Effect of antibiotic-containing extenders on Taylorella equigenitalis contaminated semen. J Equine Vet Sci 2011;31:655-660. 7. Dean CJ, Hobgood AM, Blodgett GP, et al: The addition of ticarcillin-clavulanic acid to INRA 96 extender for stallion semen cooling. Equine Vet J 2012;44:95-99. 8. Cervantes CC, Brown MP, Gronwall R, et al: Pharmacokinetics and concentrations of ceftiofur sodium in body fluids and endometrium after repeated intramuscular injections in mares. Am J Vet Res 1993;54:573-575. 9. Witte TS, Bergwerff AA, Scherpenisse P, et al. Ceftiofur derivates in serum and endometrial tissue after intramuscular administration in healthy mares. Theriogenology 2010;74:466-472. 10. Scofield D, Black J, Wittenburg L, et al: Endometrial tissue and blood plasma concentration of ceftiofur and metabolites after intramuscular administration of ceftiofur crystalline-free acid to mares. Proc Annu Conv Am Assoc Equine Pract; 2012. p. 519-520. 11. Hatzel JN, Macpherson ML, Giguere S, et al: Administration of ceftiofur crystalline-free acid to pony mares with placentitis. Proc Annu Conv Am Assoc Equine Pract; 2012. p. 521-522. 12. Ricketts SW: Treatment of equine endometritis with intrauterine irrigations of ceftiofur sodium: a comparison with mares treated in a similar manner with a mixture of sodium benzylpenicillin, neomycin sulphate, polymixin B sulphate and furaltadone hydrochloride. Pferdeheilkunde 1997;13:486-489. 13. Eilts BE, McCoy DJ, Taylor H, et al: Effect of repeated intrauterine infusions of gentamicin on the equine endometrium. Theriogenology 1988;29:1253-1259. 14. Al Bagdadi FK, Eilts BE, Richardson GF: Scanning electron microscopy of the endometrium of mares infused with gentamicin. Microsc Microanal 2004;10:280-285. 15. Bryant JE, Brown MP, Gronwall RR, et al: Study of intragastric administration of doxycycline: pharmacokinetics including body fluid, endometrial and minimum inhibitory concentrations. Equine Vet J 2000;32:233-238. 16. Ros BD, Willsallen CC, Norman ST: The renaissance of oxytetracycline-a treatment for equine endometritis? Proc Aust College Vet Sci Annu Conf 2010. p.70. 17. Papich MG, Van Camp SD, Cole JA, et al: Pharmacokinetics and endometrial tissue concentrations of enrofloxacin and the metabolite ciprofloxacin after i.v. administration of enrofloxacin to mares. J Vet Pharmacol Ther 2002;25:343-350. 18. Gonzalez C, Moreno L, Fumuso E, et al. Enrofloxacin-based therapeutic strategy for the prevention of endometritis in susceptible mares. J Vet Pharmacol Ther 2010;33:287-294. 19. Troedsson MH: Treatment strategies in mares with endometritis: Proc Soc Therio Mare Reprod Symp 1996. p. 40-50. 20. Fumuso E, Checura C, Losinno L, et al: Endometrial tissue concentrations of enrofloxacin after intrauterine administration to mares. Vet Res Commun 2002;26:371-380. 21. Rodriguez JS, Han S, Nielsen S, et al: Consequences of intrauterine enrofloxacin infusion on mare endometrium. J Equine Vet Sci 2012;32:106-111. 22. Specht TE, Brown MP, Gronwall RR, et al: Pharmacokinetics of metronidazole and its concentration in body fluids and endometrial tissues of mares. Am J Vet Res 1992;53:1807-1812. 23. Beltaire KA, Cheong SH, Coutinho da Silva MA: Retrospective study on equine uterine fungal isolates and antifungal susceptibility patterns (1999-2011). Equine Vet J 2012;44:84-87. 24. Olsen LM, Al-Bagdadi FK, Richardson GF, et al: A histological study of the effect of saline and povidone-iodine infusions on the equine endometrium. Theriogenology 1992;37:1311-1325. 25. Fuursted K, Hjort A, Knudsen L: Evaluation of bactericidal activity and lag of regrowth (postantibiotic effect) of five antiseptics on nine bacterial pathogens. J Antimicrob Chemother 1997;40:221-226. 26. Ritchie BW, Wooley RE, Kemp DT: Use of potentiated antibiotics in wound management. Vet Clin North Am Exotic Anim Pract 2004;7:169-189. 27. Nielsen JM: Endometritis in the mare: A diagnostic study comparing cultures from swab and biopsy. Theriogenology 2005;64(3):510-8. 28. Overbeck W, Witte TS, Heuwieser W: Comparison of three diagnostic methods to identify subclinical endometritis in mares. Theriogenology 2011;75:1311-1318. 29. Curras M, Magarinos B, Toranzo AE, et al: Dormancy as a survival strategy of the fish pathogen Streptococcus parauberis in the marine environment. Dis Aquat Organ 2002;52:129-136. 30. Oliver JD: Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol Rev 2010;34:415-425. 31. Keep NH, Ward JM, Cohen-Gonsaud M, et al: Wake up! Peptidoglycan lysis and bacterial non-growth states. Trends Microbiol 2006;14:271-276. 32. Kana BD, Mizrahi V: Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling. FEMS Immunol Med Microbiol 2010;58:39-50. 33. Petersen MR, Lehn-Jensen H, Bojesen AM: Use of fluorescent in situ hybridization (FISH) to identify endometritis pathogens in the mare (abstract). J Anim Sci 2010;88[E-suppl]:1097. 512Clinical Theriogenology • Volume 5, Number 4 • December 2013 34. Petersen MR, Lu KG, Christoffersen M, et al: Chronic Streptococcus equi subspecies zooepidemicus endometritis in the mare-induction of active growth and treatment improves diagnosis and fertility in the subfertile Thoroughbred mare. Proc Havemeyer Workshop Strangles and Other Streptococcal Diseases 2012. 35. Brinsko SP: Common procedures in broodmare practice: what is the evidence? Vet Clin North Am Equine Pract 2007;23:385-402. 36. Bosh KA, Powell D, Neibergs JS, et al: Impact of reproductive efficiency over time and mare financial value on economic returns among Thoroughbred mares in central Kentucky. Equine Vet J 2009;41:889-894. 37. Erol E, Locke SJ, Donahoe JK, et al: Beta-hemolytic Streptococcus spp. from horses: a retrospective study (2000- 2010). J Vet Diagn Invest 2012;24:142-147. 38. Giles RC, Donahue JM, Hong CB, et al: Causes of abortion, stillbirth, and perinatal death in horses: 3,527 cases (1986- 1991). J Am Vet Med Assoc 1993;203:1170-1175. 39. Murchie TA, Macpherson ML, LeBlanc MM, et al: Continuous monitoring of penicillin G and gentamicin in allantoic fluid of pregnant pony mares by in vivo microdialysis. Equine Vet J 2006;38:520-525. 40. Macpherson ML: Treatment strategies for mares with placentitis. Theriogenology 2005;64:528-534. 41. Bailey CS, Macpherson ML, Pozor MA, et al. Treatment efficacy of trimethoprim sulfamethoxazole, pentoxifylline and altrenogest in experimentally induced equine placentitis. Theriogenology 2010;74:402-412. 513 Clinical Theriogenology • Volume 5, Number 4 • December 2013 Table 1. Antimicrobial routes and doses Antimicrobial Systemic Dose Intrauterine Dose Procaine penicillin G 22,000 IU/kg 5 million IU Potassium penicillin 22,000 IU/kg 5 million IU Ampicillin 20-40 mg/kg IV TID- QID 3 g Ticarcillin and clavulanic acid 50-60 mg/kg IV TID- QID 3.1-6.2 g 0.5 to 1.5 mg/mL in Inra96 Ceftiofur sodium 2.2 mg/kg IV/IM SID- BID 1 g Marketed as Naxcel in US Ceftiofur crystalline free acid 6.6 mg/kg IM 2 doses 4 days apart Marketed as Excede in US Ceftiofur hydrochloride 1 g Marketed as Excenel in US Polymyxin B 6000 U/kg IV BID- TID (antiendotoxic) 1 million U Amikacin 10-15 mg/kg IV SID (adults) 2 g Gentamicin 6.6 mg/kg IV SID 500-2000 mg Buffered or diluted Oxytetracycline 6.6-10 mg/kg SID- BID slow dilute IV (6 g)* *pending further study Doxycycline 10 mg/kg PO BID Enrofloxacin 6 mg/kg IV SID Ciprofloxacin 2.5 g PO SID with 1 g Probenecid PO SID Amphotericin B 0.3-0.5 mg/kg IV EOD 100-200 mg Nystatin 0.5-2.5 million U Ketoconazole 10 mg/kg in 0.2N HCl NGtube BID Clotrimazole 500-700 mg Miconazole 400-700 mg Itraconazole 6 mg/kg PO SID Fluconazole 5 mg/kg PO SID 100 mg 514Clinical Theriogenology • Volume 5, Number 4 • December 2013 T ab le 2 . S en si ti vi ty o f ut er in e or ga ni sm s is ol at ed a t H ag ya rd L ab or to ry in 2 01 2. R es ul ts a re b as ed o n cu lt ur es c ol le ct ed b y H ag ya rd v et er in ar ia ns in C en tr al K en tu ck y. T ot al n um be r of e nd om et ri al c ul tu re s= 60 62 . * - A nt ib io ti c no t r ec om m en de d fo r us e w it h th is o rg an is m p er C li ni ca l L ab or at or y S ta nd ar ds I ns ti tu te ( C L S I) G ui de li ne s. P en ic il li n su sc ep ti bl e be ta S tr ep to co cc us s pe ci es c an b e co ns id er ed s us ce pt ib le to a m pi ci ll in , ce fa zo li n an d im ip en em p er C L S I G ui de li ne s. O f th e 60 62 c ul tu re s, 3 0 ye as t s pe ci es a nd 1 7 fu ng al s pe ci es w er e id en ti fi ed . 515 Clinical Theriogenology • Volume 5, Number 4 • December 2013 516Clinical Theriogenology • Volume 5, Number 4 • December 2013 OMNIBLANK: